BACKGROUND OF THE INVENTION
[0001] The immune system must be able to discriminate between self and non-self. When self/non-self
discrimination fails, the immune system destroys cells and tissues of the body and
as a result causes autoimmune diseases. Regulatory T cells actively suppress activation
of the immune system and prevent pathological self-reactivity and consequent autoimmune
disease. Developing drugs and methods to selectively activate regulatory T cells for
the treatment of autoimmune disease is the subject of intense research and, until
the development of the present invention, has been largely unsuccessful.
[0002] Regulatory T cells (Treg) are a class of CD4+CD25+ T cells that suppress the activity
of other immune cells. Treg are central to immune system homeostasis, and play a major
role in maintaining tolerance to self-antigens and in modulating the immune response
to foreign antigens. Multiple autoimmune and inflammatory diseases, including Type
1 Diabetes (T1D), Systemic Lupus Erythematosus (SLE), and Graft-versus-Host Disease
(GVHD) have been shown to have a deficiency of Treg cell numbers or Treg function.
Consequently, there is great interest in the development of therapies that boost the
numbers and/or function of Treg cells.
[0003] One treatment approach for autoimmune diseases being investigated is the transplantation
of autologous,
ex vivo-expanded Treg cells (
Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in treating animal models of disease and
in several early stage human clinical trials, it requires personalized treatment with
the patient's own T cells, is invasive, and is technically complex. Another approach
is treatment with low dose Interleukin-2 (IL-2). Treg cells characteristically express
high constitutive levels of the high affinity IL-2 receptor, IL2Pαβγ, which is composed
of the subunits IL2RA (CD25), IL2RB (CD122), and IL2RG (CD132), and Treg cell growth
has been shown to be dependent on IL-2 (
Malek T. R., et al., 2010, Immunity, 33: 153-65). Clinical trials of low-dose IL-2 treatment of chronic GVHD (
Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis patients (
Saadoum, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical
trials investigating the efficacy of IL-2 in multiple other autoimmune and inflammatory
diseases have been initiated.
[0004] Proleukin (marketed by Prometheus Laboratories, San Diego, CA), the recombinant form
of IL-2 used in these trials, is associated with high toxicity. Proleukin is approved
for the treatment of Metastatic Melanoma and Metastatic Renal Cancer, but its side
effects are so severe that its use is only recommended in a hospital setting with
access to intensive care (http://www.proleukin.com/assets/pdf/proleukin.pdf). Until
the more recent characterization of of Treg cells, IL-2 was considered to be immune
system stimulator, activating T cells and other immune cells to eliminate cancer cells.
The clinical trials of IL-2 in autoimmune diseases have employed lower doses of IL-2
in order to target Treg cells, because Treg cells respond to lower concentrations
of IL-2 than many other immune cell types because of their expression of IL2Rαβγ (
Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower doses resulted in safety and tolerability issues, and
the treatments used have employed daily subcutaneous injections, either chronically
or in intermittent 5 day treatment courses. Therefore, there is need for an autoimmune
disease therapy that potentiates Treg cell numbers and function, that targets Treg
cells more specifically than IL-2, that is safer and more tolerable, and that is administered
less frequently.
[0005] One approach to improving the therapeutic index of IL-2-based therapy is to use variants
of IL-2 that are selective for Treg cells relative to other immune cells. IL-2 receptors
are expressed on a variety of different immune cell types, including T cells, NK cells,
eosinophils, and monocytes, and this broad expression pattern likely contributes to
its pleiotropic effect on the immune system and high systemic toxicity. The IL-2 receptor
exists in three forms: (1) the low affinity receptor, IL2RA, which does not signal;
(2) the intermediate affinity receptor (IL2Rβγ), composed of IL2RB and IL2RG, which
is broadly expressed on conventional T cells (Tcons), NK cells, eosinophils, and monocytes;
and (3) the high affinity receptor (IL2Rαβγ), composed of IL2RA, IL2RB, and IL2RG,
which is expressed transiently on activated T cells and constitutively on Treg cells.
IL-2 variants have been developed that are selective for IL2Rαβγ relative to IL2Rβγ
(
Shanafelt, A. B., et al., 2000, Nat Biotechnol. 18:1197-202;
Cassell, D. J., et al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions which reduce their affinity for IL2RB.
Because IL-2 has undetectable affinity for IL2RG, these variants consequently have
reduced affinity for the IL2Rβγ receptor complex and reduced ability to activate IL2Rβγ-expressing
cells, but retain the ability to bind IL2RA and the ability to bind and activate the
IL2Rαβγ receptor complex. One of these variants, IL2/N88R (Bay 50-4798), was clinically
tested as a low-toxicity version of IL-2 as an immune system stimulator, based on
the hypothesis that IL2Rβγ-expressing NK cells are a major contributor to toxicity.
Bay 50-4798 was shown to selectively stimulate the proliferation of activated T cells
relative to NK cells, and was evaluated in phase I/II clinical trials in cancer patients
(
Margolin, K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (
Davey, R. T., et. al., 2008, J Interferon Cytokine Res., 28:89-100). These trials showed that Bay 50-4798 was considerably safer and more tolerable
than Proleukin, and also showed that it increased the levels of CD4+ T cells and CD4+CD25+
T cells in patients. However, the increase in CD4+ T cells and CD4+CD25+ T cells were
not indicative of an increase in Treg cells, because identification of Tregs requires
additional markers in addition to CD4 and CD25, and because Treg cells are a minor
fraction of CD4+CD25+ cells. Subsequent to these trials, research in the field more
fully established the identity of Treg cells and demonstrated that Treg cells selectively
express IL2Rαβγ (reviewed in
Malek, T. R., et al., 2010, Immunity, 33:153-65). Based on this new research, it can now be understood that IL2Rαβγ selective agonists
should be selective for Treg cells.
[0006] A second approach to improving the therapeutic index of an IL-2 based therapy is
to optimize the pharmacokinetics of the molecule to maximally stimulate Treg cells.
Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation
in vitro required a minimum of 5-6 hours exposure to effective concentrations of IL-2 (
Cantrell, D. A., et. al., 1984, Science, 224: 1312-1316). When administered to human patients, IL-2 has a very short plasma half-life of
85 minutes for intravenous administration and 3.3 hours subcutaneous administration
(
Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Because of its short half-life, maintaining circulating IL-2 at or above the level
necessary to stimulate T cell proliferation for the necessary duration necessitates
high doses that result in peak IL-2 levels significantly above the EC50 for Treg cells
or will require frequent administration (FIGURE 1). These high IL-2 peak levels can
activate IL2Rβγ receptors and have other unintended or adverse effects. An IL-2 analog
with a longer circulating half-life than IL-2 can achieve a target drug concentration
for a specified period of time at a lower dose than IL-2, and with lower peak levels.
Such an IL-2 analog will therefore require either lower doses or less frequent administration
than IL-2 to effectively stimulate Treg cells. Indeed, in cynomolgus monkeys dosed
with an IgG-IL2 fusion protein with a circulating half-life of 14 hours stimulated
a much more robust increase in Tregs compared to an equimolar dose of IL-2 (
Bell, et al., 2015, J Autoimmun. 56:66-80). Less frequent subcutaneous administration of an IL-2 drug will also be more tolerable
for patients. A therapeutic with these characteristics will translate clinically into
improved pharmacological efficacy, reduced toxicity, and improved patient compliance
with therapy.
[0007] One approach to extending the half-life of therapeutic proteins is to fuse the therapeutically
active portion of the molecule to another protein, such as the Fc region of IgG, to
increase the circulating half-life. Fusion of therapeutic proteins with IgG Fc accomplishes
this by increasing the hydrodynamic radius of the protein, thus reducing renal clearance,
and through Neonatal Fc Receptor (FcRn)-mediated recycling of the fusion protein,
thus prolonging the circulating half-life. The fusion of therapeutic proteins to albumin
(
Sleep, D., et. al., 2013, Biochem Biophys Acta., 1830:5526-34) and nonimmunogenic amino acid polymer proteins (
Schlapschy, M., et. al., 2007, Protein Eng Des Sel. 20:273-84;
Schellenberger, V., et al., 2009, Nat Biotechnol. 27:1186-90) have also been employed to increase circulating half-life. However, construction
of such fusion proteins in a manner that ensures robust biological activity of the
IL2 Selective Agonist fusion partner can be unpredictable, especially in the case
of an IL-2 Selective Agonist, which is a small protein that is defective in binding
to one of the receptor subunits and that must assemble a complex of three receptor
subunits in order to activate the receptor (
Wang, X., et al., 2005, Science 310:1159-63).
[0008] Other researchers have created various IL-2 fusion proteins, using wild-type IL-2
or IL-2 with a C125S substitution to promote stability. Morrison and colleagues (
Penichet, M. L., et, al.,1997, Hum Antibodies. 8:106-18) created a fusion protein with IgG fused to wild-type IL-2 to both increase the circulating
half-life of IL-2 and to target IL-2 to specific antigens for the purpose of potentiating
the immune response to the antigen. This fusion protein consisted of an intact antibody
molecule, composed of heavy (H) and light (L) chains, wherein the N-terminal H chain
moiety was fused to a C-terminal IL-2 protein moiety. This IgG-IL-2 fusion protein
possessed Fc effector functions. Key effector functions of IgG Fc proteins are Complement-dependent
cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The IgG-IL-2
fusion protein was highly active in an IL-2 bioassay and was shown to possess CDC
activity. Thus, Penichet et. al. taught the use of antibody-IL2 fusion proteins to
target IL-2 activity to antigens recognized by the antibody, for the purpose of potentiating
humoral and cell-mediated immune responses to the antigen. In a similar manner, Gillies
and colleagues have constructed a number of IgG-IL-2 fusion proteins for cancer immunotherapy,
utilizing the antibody portion of the fusion protein to target tumor antigens, and
the IL-2 portion to stimulate the immune response to tumor cells (reviewed in
Sondel, P. M., et al., 2012, Antibodies, 1:149-71).
WO 2008/003473 describes compositions and methods for enhancing the efficacy of IL-2 mediated immune
responses. These teachings are quite distinct from the present inventive technology,
wherein an IL-2 selective agonist, which promotes the growth and activity of immunosuppressive
Treg cells, is fused with an effector function-deficient Fc protein moiety for the
purpose increasing systemic exposure.
[0009] Strom and his colleagues have constructed fusion proteins with IL-2 fused to the
N terminus of an Fc protein for the purpose of eliminating activating T cells expressing
the high-affinity IL-2 receptor (
Zheng, X. X., et al., 1999, J Immunol. 1999, 163:4041-8). This fusion protein was shown to inhibit the development of autoimmune diabetes
in a T cell transfer mouse model of T1D. The IL2-Fc fusion protein was shown to inhibit
the function of disease-promoting T cells from TID-susceptible female NOD mice when
transplanted into less disease-susceptible male NOD mice. They also demonstrated that
the IL-2-Fc fusion protein could kill cells expressing the high-affinity IL-2 receptor
in vitro. These investigators further compared IL2-Fc fusion proteins constructed from an Fc
derived from an effector function-competent IgG2b Fc and a mutated effector function-deficient
IgG2b Fc. Only the IL2-Fc fusion protein containing the effector function-competent
Fc was efficacious in preventing disease onset. Thus, these investigators teach that
an IL2-Fc fusion protein with effector functions can eliminate disease-causing activated
T cells, and that Fc effector functions are necessary for its therapeutic activity.
These teachings are quite distinct from the present inventive technology, wherein
an IL-2 selective agonist, which promotes the growth and activity of immunosuppressive
Treg cells, is fused with an effector function-deficient Fc protein moiety for the
purpose increasing systemic exposure and optimizing Treg expansion. Other work from
Strom and colleagues teaches the use of a IL2-Fc fusion protein in promoting transplant
tolerance (
Zheng, X, X., et al., 2003, Immunity, 19:503-14). In this work, an IL2-Fc fusion protein is used in a "triple therapy" in which it
is combined with an IL15-Fc receptor antagonist and rapamycin. Again, these investigators
teach that the IL2-Fc fusion protein must have Fc effector functions to be efficacious,
and further teach that this IL-2-Fc fusion protein must be combined with two other
molecules in order to be efficacious.
[0010] This invention provides for a novel therapeutic agent, an IL2 Selective Agonist-
Fc fusion protein, that combines the high cellular selectivity of a IL2 Selective
Agonist for Treg cells with a long circulating half-life. In the course of developing
this molecule, there were surprising and unexpected findings that revealed structural
elements and design features of the protein that are essential for bioactivity, and
that led to the discovery of several novel proteins that fulfill the desired therapeutic
characteristics.
BRIEF SUMMARY OF THE INVENTION
[0011] The invention provides a fusion protein, comprising: a N-terminal human IL-2 variant
protein having at least 95% sequence identity to SEQ ID NO: 1 and comprising the substitution
C125S and a substitution selected from the group consisting of: N88R, N88I, N88G,
D20H, Q126L and Q126F; a C-terminal IgG Fc protein; and a linker peptide of between
5 to 20 amino acid residues positioned between the IL-2 variant protein and the IgG
Fc protein. The fusion protein is between an IE2Rαβγ Selective Agonist protein (IL2
Selective Agonist) and a IgG Fc protein. The IL2 Selective Agonist moiety provides
a therapeutic activity by selectively activating the IE2Rαβγ form of the receptor,
thus selectively stimulating Tregs. The Fc moiety provides a prolonged circulating
half-life compared to the circulating half-life of IL-2 or an IL2 Selective Agonist
protein. The Fc moiety increases circulating half-life by increasing the molecular
size of the fusion protein to greater than 60,000 daltons, which is the approximate
cutoff for glomerular filtration of macromolecules by the kidney, and by recycling
the fusion protein through the Neonatal Fc Receptor (FcRn) protein, the receptor that
binds and recycles IgG, thus prolonging its circulating half-life. The Fc moiety will
also be deficient in Fc effector functions, such as Complement-Dependent Cytotoxicity
(CDC) and Antibody-Dependent Cellular Cytotoxicity (ADCC), enabling the fusion protein
to selectively activate Tregs to potentiate Treg function and to expand Treg numbers.
The two protein moieties are fused in a manner that maintains robust bioactivity of
the IL2 Selective Agonist moiety and enables the Fc moiety to promote a prolonged
circulating half-life and thus efficiently potentiate Tregs function and numbers.
This potentiation of Tregs will suppress over-exuberant autoimmune or inflammatory
responses, and will be of benefit in treating autoimmune and inflammatory diseases.
The proteins of this invention may be monomeric or dimeric forming dimers through
cysteine residues in the Fc moieties or domains.
[0012] More specifically, this invention provides for a fusion protein, comprising: a N-terminal
human IL-2 variant protein moiety, and a C-terminal IgG Fc protein moiety, wherein
said IL-2 fusion protein has the ability to selectively activate the high affinity
IL-2 receptor and thus selectively activate human regulatory T cells. The variants
of IL-2 include those with substitutions selected from the group consisting of: N88R,
N88I, N88G, D20H, Q126L, and Q126F relative to human IL2 protein (SEQ ID NO:1). In
addition the, IL-2 variant protein comprises human IL-2 with the substitution C125S.
The proteins of this invention are fused wherein both the IL-2 variant protein and
the IgG Fc protein have an N-terminus and a C-terminus and said human IL-2 variant
protein is fused at its C-terminus to the N-terminus of the IgG Fc protein. The IL-2
variant domain and the Fc domain are joined or fused through a linker peptide positioned
between the IL-2 variant protein and the IgG Fc protein moieties. The IgG Fc protein
moiety or domain will preferably be deficient in Fc effector functions or contain
one or more amino acid substitutions that reduce the effector functions of the Fc
portion of the fusion protein.
[0013] An example of this invention is a protein, comprising: a IL-2 variant protein having
amino acid substitutions N88R and C125S relative to human IL-2 (SEQ IID NO:1), a linker
peptide as set forth in SEQ ID NO: 15, and a human IgG1 Fc protein as set forth in
SEQ ID NO:2, wherein said fusion protein has the ability to selectively activate the
high affinity IL-2 receptor and thus selectively activate human regulatory T cells.
Alternative proteins of this invention include: a IL-2 variant protein having amino
add substitutions N88R and C125S relative to human IL-2 (SEQ IID NO: 1), a linker
peptide as set forth in SEQ ID NO:15, and a human IgG2 Fc protein as set forth in
SEQ ID NO:3.
[0014] A more specific embodiment of this invention is a dimeric protein, comprising two
identical chains, where each chain comprises a N-terminal human IL-2 variant protein
moiety and a C-terminal IgG Fc protein moiety wherein: the N-terminal human IL-2 variant
protein moiety has a N-terminus and a C- terminus varies from the human IL-2 wildtype
as in SEQ ID NO:1 by at least one of the substitutions selected from the group consisting
of: N88R, N88I, N88G, D20H, Q126L, and Q126F, has at least a 97% sequence identify
to Sequence ID No. 1; and, has the ability to activate Treg cells by binding to a
IL2Rαβγ on those cells; the N-terminal human IL-2 variant protein is joined at its
C-terminal to a N-terminus of an amino acid linker of between 6 to 20 amino acid residues
where said linker also has a C-terminus; and, the C-terminus of the amino acid linker
is joined to the N-terminus of IgG Fc protein moiety having 97% sequence identify
to for example SEQ ID NO:3 (IgG2) or SEQ ID No. 2 (IgG1N297A) and containing cysteine
residues; and where the two chains are linked to each other through the cysteine residues
that form the interchain disulfide bonds of the IgG Fc protein moiety. The dimers
of this invention are further substituted at C125S of the IL-2 moiety. The proteins
of this invention include amino acid linkers, preferably consisting a group of glycine
residues, serine residues, and a mix of glycine and serine residues. The linkers may
comprise a mix of between 12 and 17 serine and glycine residues preferably with a
ratio of glycine residues to serine residues in a range of 3:1-5:1, e.g, a 4:1 ratio.
[0015] This invention further provides for the compositions above in a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
[0016] This invention further provides for nucleic acids encoding the proteins described
herein. The nucleic acids or preferably operably linked to expression cassettes that
can be either designed for recombination with a host cell genome or introduced on
an independently replicating plasmid or extrachromosomal nucleic acid.
[0017] Further disclosed are methods of selectively activating human regulatory T cells
in a patient in need thereof, the method comprising administering a pharmaceutical
composition comprising the compositions described administered at therapeutically
effective doses until human regulatory T cell concentrations reach desired levels.
[0018] A method of measuring the numbers of Treg cells in a human blood sample by contacting
human blood cells with the fusion protein of claim 1 at a concentration of between
1 nM and 0.01 nM, and then detecting cells that bind to the protein by flow cytometry
is also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019]
FIGURE 1 is a diagrammatic illustration of the relationship between circulating half-life,
peak drug level, the biological effective concentration, and the duration necessary
to stimulate Treg cell proliferation after a single dose of IL-2 or an IL2-Fc fusion
protein with increased half-life. The dashed line represents the blood level over
time of IL-2 following a subcutaneous injection, and the solid line represents the
blood level over time of an IL2-Fc fusion protein. The horizontal dotted lines indicate
the concentrations (EC50 values) necessary to activate cells expressing IL2Rαβγ and
IL2Rβγ, respectively) are indicated. The double-headed arrow indicates the duration
of exposure (5-6 hr) to IL-2 at the EC50 necessary to stimulate cell proliferation.
FIGURE 2 shows the design configurations for Fc fusion proteins. The fusion partner
(X), can be fused at the N terminus (X-Fc) or the C-terminus (Fc-X) of the Fc protein.
Linker peptides can be inserted between X and the Fc.
FIGURE 3 shows a dose-response of IL-2 and N88RL9AG1 stimulated STAT5 phosphorylation
in CD4+ T cells as measured by flow cytometry. Cells were treated with the IL-2 or
N88RFc at the concentrations indicated on the top for 10 minutes at 37 C, fixed, permeabilized,
stained with antibodies, and then subjected to flow cytometry analysis as described
in Example 3. Cells gated as CD4+ are shown, and cells further gated with respect
to CD25 and pSTAT5 as shown in each of the 4 quadrants. The numbers in each quadrant
indicate the percentage of CD4+ cells in each gate. Cells in the upper quadrants represent
the highest 1-2% of CD25 expressing cells, a population enriched for Treg cells, and
cells in the right-hand quadrants are pSTAT5+. A. N88RL9AG1 stimulates only CD25high cells with high selectivity, while IL-2 massively stimulates both CD25-/low and CD25high cells down to picomolar concentrations. B. D20HL0G2 has no pSTAT5 stimulating activity.
No pSTAT5 activation was observed in two independent experiments. C. Control showing
that D20H/IL2 stimulates pSTAT5 in CD25high cells while D20HL0G2 does not. Plots are displayed in the pseudocolor mode. Both
proteins were tested at a concentration of 10-8 M.
FIGURE 4 shows that CD4+ T cells treated with N88RL9AG1 exhibited stimulation of pSTAT5
levels in cells expressing high levels of FOXP3. Cells were treated with 4 X 10-9 M IL-2 or N88RL9AG1 and then analyzed as described in Example 3. The majority of
pSTAT5+ cells treated with N88RL9AG1 were also FOXP3+, whereas pSTAT5+ cells treated
with IL-2 were both FOXP3- and FOXP3+, with the majority being FOXP3-.
FIGURE 5 shows the protein yields of different Fc fusion constructs produced in HEK293
cells. Proteins were expressed in parallel in an optimized transient expression system
and purified as described in Example 1. Results are expressed as the final yield of
purified protein from 30 ml cultures. A. Protein yields of N88R/IL2-Fc fusion proteins
increase with increasing peptide linker length. B. Yields of wt IL2-Fe fusion proteins are only slightly enhanced with a 15 residue peptide linker.
Higher yields of D20H/IL2-Fc fusion proteins were obtained in the X-Fc rather than
the Fc-X configuration.
FIGURE 6 shows the dependence of IL-2 bioactivity on peptide linker length in N88R/IL2-Fc
fusion proteins. (A) pSTAT5 signals in CD25high CD4+ T cells (Tregs) increase with increasing peptide linker length. (B) No significant
pSTAT5 signal with any of N88R/IL2-Fc proteins was observed in CD25-/low cells. The pSTAT5 signal of the 10-8 M IL-2 internal control is indicated in both panels by the black triangle.
FIGURE 7 shows the bioactivity of D20H/IL2-Fc fusion proteins in human Tregs. The
potency of D20HL15AG1 is substantially less than that of N88RL15AG1, and D20HL15AG1
(X-Fc configuration) and AG1L15D20H (Fc-X configuration) have similar potencies. All
3 proteins have a 15 residue peptide linker.
FIGURE 8 shows the bioactivity of wt IL-2-Fc pSTAT5 activity with and without a 15 residue peptide linker. IL-2 bioactivity
is only modestly enhanced by a 15 residue peptide linker in both Tregs (A) and in
CD25-/low cells (B).
FIGURE 9. Selectivity of IL-2 and IL-2 Selective Agonist proteins on 7 different immune
cell types in human PBMC. N88RL15AG1 is highly selectivity for Tregs compared to wt IL-2 and WTL15AG1, and shows greater selectivity in multiple cell types than N88R/IL2.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0020] This invention is a novel therapeutic fusion protein that comprises three key protein
elements: (1) an engineered IL-2 cytokine that has been modified to be highly selective
for Treg cells, (2) an effector function deficient Fc protein that will increase the
circulating half-life of the protein, and (3) a linker peptide between the two moieties
that is necessary for high biological activity of the fusion protein. The fusion proteins
which constitute this invention were discovered through initial unanticipated findings
that went against teachings in the prior art of IL-2 fusion proteins, and the research
that led to these molecules has defined key structure-activity relationships important
for their biological and therapeutic activity. The molecules defined by this invention
will enable the safe and effective treatment of autoimmune diseases by the novel mechanism
of stimulating the production of a small subpopulation of T cells that suppress autoimmune
and inflammatory pathology. This paradigm-breaking therapeutic can potentially treat
a number of different autoimmune diseases.
Definitions
[0021] "At least a percent (eg. 97%) sequence identify to Sequence ID No. 1" as used herein
refers to the extent to which the sequence of two or more nucleic acids or polypeptides
is the same. The percent identity between a sequence of interest and a second sequence
over a window of evaluation, e.g., over the length of the sequence of interest, may
be computed by aligning the sequences, determining the number of residues (nucleotides
or amino acids) within the window of evaluation that are opposite an identical residue
allowing the introduction of gaps to maximize identity, dividing by the total number
of residues of the sequence of interest or the second sequence (whichever is greater)
that fall within the window, and multiplying by 100. When computing the number of
identical residues needed to achieve a particular percent identity, fractions are
to be rounded to the nearest whole number. Percent identity can be calculated with
the use of a variety of computer programs. For example, computer programs such as
BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent
identity between sequences of interest. The algorithm of Karlin and Altschul (
Karlin and Altschul, Proc. Natl. Acad. ScL USA 87:22264-2268, 1990) modified as in
Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (
Altschul, et al., J. Mol. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as
described in Altschul et al. (
Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective
programs may be used. A PAM250 or BLOSUM62 matrix may be used. Software for performing
BLAST analyses is publicly available through the National Center for Biotechnology
Information (NCBI). See the Web site having URL world-wide web address of: "ncbi.nlm.nih.gov"
for these programs. In a specific embodiment, percent identity is calculated using
BLAST2 with default parameters as provided by the NCBI.
[0022] "N-terminus" refers to the end of a peptide or polypeptide that bears an amino group
in contrast to the carboxyl end bearing a carboxyl acid group.
[0023] "C- terminus" refers to the end of a peptide or polypeptide that bears a carboxcylic
acid group in contrast to the amino terminus bearing an amino group.
[0024] "C-terminal IgG Fc protein moiety" refers to a portion of a fusion protein that derives
from two identical protein fragments, each having a hinge region, a second constant
domain, and a third constant domains of the IgG molecule's two heavy chains, and consisting
of the carboxy-terminal heavy chains disulphide bonded to each other through the hinge
region. It is functionally defined as that part of the IgG molecule that interacts
with the complement protein C1q and the IgG-Fc receptors (FcγR), mediating Complement-dependent
cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) effector functions.
The sequence can be modified to decrease effector functions, to increase circulating
half-life, and to eliminate glycoslylation sites.
IL2 variants
[0025] IL-2 variant proteins of this invention are IL-2αβγ Selective Agonists. Functionally
they selectively activate the IL2Rαβγ receptor complex relative to the IL2Rβγ receptor
complex. It is derived from a wild type IL-2 protein structurally defined as having
at least a 95% sequence identity to the wild type IL-2 of Sequence ID No. 1 and functionally
defined by the ability to preferentially activate Treg cells. The protein can also
be functionally defined by its ability to selectively activate IL-2 receptor signaling
in Tregs, as measured by the levels of phosphorylated STAT5 protein in Treg cells
compared to CD4+ CD25-/low T cells or NK cells, or by the selective activation of
Phytohemagglutinin-stimulated T cells versus NK cells.
[0026] "N-terminal human IL-2 variant protein moiety" refers to a N-terminal domain of a
fusion protein that is derived from a wild type IL-2 protein structurally and functionally
defines above.
[0027] "C- terminus" refers to the end of a peptide or polypeptide that bears a carboxcylic
acid group in contrast to the amino terminus bearing an amino group.
Tregs
[0028] "Tregs" or "Treg cells" refer to Regulatory T cells. Regulatory T cells are a class
of T cells that suppress the activity of other immune cells, and are defined using
flow cytometry by the cell marker phenotype CD4+CD25+FOXP3+. Because FOXP3 is an intracellular
protein and requires cell fixation and permeablization for staining, the cell surface
phenotype CD4+CD25+CD127- can be used for defining live Tregs. Tregs also include
various Treg subclasses, such as tTregs (thymus-derived) and pTregs (peripherally-derived,
differentiated from naive T cells in the periphery). All Tregs express the IL2Rαβγ
receptor, do not produce their own IL-2 and are dependent on IL-2 for growth, and
someone skilled in the art will recognize that both classes will be selectively activated
by a IL2Rαβγ selective agonist
Peptide Linkers
[0029] "Peptide linker" is defined as an amino acid sequence located between the two proteins
comprising a fusion protein, such that the linker peptide sequence is not derived
from either partner protein. Peptide linkers are incorporated into fusion proteins
as spacers in order to promote proper protein folding and stability of the component
protein moieties, to improve protein expression, or to enable better bioactivity of
the two fusion partners (
Chen, et al., 2013, Adv Drug Deliv Rev. 65(10):1357-69). Peptide linkers can be divided into the categories of unstructured flexible peptides
or rigid structured peptides.
Fc fusion proteins
[0030] An "Fc fusion protein" is a protein made by recombinant DNA technology in which the
translational reading frame of the Fc domain of a mammalian IgG protein is fused to
that of another protein ("Fc fusion partner") to produce a novel single recombinant
polypeptide. Fc fusion proteins are typically produced as disulfide-linked dimers,
joined together by disulfide bonds located in the hinge region.
Functional activation
[0031] "Bioactivity" refers to the measurement of biological activity in a quantitative
cell-based
in vitro assay.
[0032] "Functional activation of Treg cells" is defined an IL-2-mediated response in Tregs.
Assay readouts for functional activation of Treg cells includes stimulation of pSTAT5,
Treg cell proliferation, and stimulation of the levels of Treg effector proteins.
DESIGN AND CONSTRUCTION
[0033] There are multiple options for the design and construction of an Fc fusion protein,
and the choices among these design options are presented below to permit the generation
of a molecule with the desired biological activity and pharmaceutical characteristics.
Key design options are: (1) the nature of the IL2 Selective Agonist, (2) the choice
of the Fc protein moiety, (3) the configuration of fusion partners in the fusion protein,
and (4) the amino acid sequence at the junction between the Fc and the fusion partner
protein.
General Methods
[0034] In general, preparation of the fusion proteins of the invention can be accomplished
by procedures disclosed herein and by recognized recombinant DNA techniques involving,
e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA,
cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation
of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation
or transfection of a host, culturing of the host. Additionally, the fusion molecules
can be isolated and purified using chaotropic agents and well known electrophoretic,
centrifugation and chromatographic methods. See generally,
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York
(1989) for disclosure relating to these methods.
[0035] The genes encoding the fusion proteins of this invention involve restriction enzyme
digestion and ligation as the basic steps employed to yield DNA encoding the desired
fusions. The ends of the DNA fragment may require modification prior to ligation,
and this may be accomplished by filling in overhangs, deleting terminal portions of
the fragment(s) with nucleases (e.g., ExoIII), site directed mutagenesis, or by adding
new base pairs by PCR. Polylinkers and adaptors may be employed to facilitate joining
of selected fragments. The expression construct is typically assembled in stages employing
rounds of restriction, ligation, and transformation of E. coli. Numerous cloning vectors
suitable for construction of the expression construct are known in the art (lambda.ZAP
and pBLUESCRIPT SK-1, Stratagene, LaJolla, Calif., pET, Novagen Inc., Madison, W is.--cited
in Ausubel et al., 1999) and the particular choice is not critical to the invention.
The selection of cloning vector will be influenced by the gene transfer system selected
for introduction of the expression construct into the host cell. At the end of each
stage, the resulting construct may be analyzed by restriction, DNA sequence, hybridization
and PCR analyses.
[0037] Various promoters (transcriptional initiation regulatory region) may be used according
to the invention. The selection of the appropriate promoter is dependent upon the
proposed expression host. Promoters from heterologous sources may be used as long
as they are functional in the chosen host
[0038] Various signal sequences may be used to facilitate expression of the proteins described
herein. Signal sequence are selected or designed for efficient secretion and processing
in the expression host may also be used. A signal sequence which is homologous to
the TCR coding sequence or the mouse IL-2 coding sequence may be used for mammalian
cells. Other suitable signal sequence/host cell pairs include the
B. subtilis sacB signal sequence for secretion in B. subtilis, and the
Saccharomyces cerevisiae α-mating factor or
P. pastoris acid phosphatase phoI signal sequences for
P.
pastoris secretion. The signal sequence may be joined directly through the sequence encoding
the signal peptidase cleavage site to the protein coding sequence, or through a short
nucleotide bridge.
[0039] Elements for enhancing transcription and translation have been identified for eukaryotic
protein expression systems. For example, positioning the cauliflower mosaic virus
(CaMV) promoter 1000 bp on either side of a heterologous promoter may elevate transcriptional
levels by 10- to 400-fold in plant cells. The expression construct should also include
the appropriate translational initiation sequences. Modification of the expression
construct to include a Kozak consensus sequence for proper translational initiation
may increase the level of translation by 10 fold.
[0040] The expression cassettes are joined to appropriate vectors compatible with the host
that is being employed. The vector must be able to accommodate the DNA sequence coding
for the fusion proteins to be expressed. Suitable host cells include eukaryotic and
prokaryotic cells, preferably those cells that can be easily transformed and exhibit
rapid growth in culture medium. Specifically preferred hosts cells include prokaryotes
such as E. coli, Bacillus subtillus, etc. and eukaryotes such as animal cells and
yeast strains, e.g., S. cerevisiae. Mammalian cells are generally preferred, particularly
HEK, J558, NSO, SP2-O or CHO. Other suitable hosts include, e.g., insect cells such
as Sf9. Conventional culturing conditions are employed. See Sambrook, supra. Stable
transformed or transfected cell lines can then be selected. In vitro transcription-translation
systems can also be employed as an expression system.
[0041] Nucleic acid encoding a desired fusion protein can be introduced into a host cell
by standard techniques for transfecting cells. The term "transfecting" or "transfection"
is intended to encompass all conventional techniques for introducing nucleic acid
into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated
transfection, lipofection, electroporation, microinjection, viral transduction and/or
integration. Suitable methods for transfecting host cells can be found in Sambrook
et al. supra, and other laboratory textbooks.
[0042] Alternatively, one can use synthetic gene construction for all or part of the construction
of the proteins described herein. This entails
in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide molecule
of interest. Gene synthesis can be performed utilizing a number of techniques, such
as the multiplex microchip-based technology described by Tian, et. al., (
Tian, et. al., Nature 432:1050-1054) and similar technologies wherein olgionucleotides are synthesized and assembled
upon photo-programmable microfluidic chips.
[0043] The fusion proteins of this invention are isolated from harvested host cells or from
the culture medium. Standard protein purification techniques are used to isolate the
proteins of interest from the medium or from the harvested cells. In particular, the
purification techniques can be used to express and purify a desired fusion protein
on a large-scale (i.e. in at least milligram quantities) from a variety of approaches
including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a
fermentor.
THE IL2 SELECTIVE AGONIST MOIETY
[0044] IL-2 with the substitution N88R is an exemplary case of an IL2 Selective Agonist
for the IL2Rαβγ receptor (
Shanafelt, A. B., et al., 2000, Nat Biotechnol.18:1197-202). IL2/N88R is deficient in binding to the IL2Rβ receptor subunit and the IL2Rβγ receptor
complex, but is able to bind to the IL2Rαβγ receptor complex and stimulate the proliferation
of IL2Rαβγ -expressing PHA-activated T cells as effectively as
wt IL-2, while exhibiting a 3,000 fold reduced ability to stimulate the proliferation
of IL2Rβγ-expressing NK cells, Other IL2Rαβγ selective agonists with similar activity
profiles include IL-2 with the substitutions N88I, N88G, and D20H, and other IL2 variants
with the substitutions Q126L and Q126F (contact residues with the IL2RG subunit) also
possess IL2Rαβγ -selective agonist activity (
Cassell, D. J., et. al., 2002, Curr Pharm Des., 8:2171-83). A practitioner skilled in the art would recognize that any of these IL2 Selective
Agonist molecules can be substituted for the IL2/N88R moiety with the expectation
that an Fc fusion protein will have similar activity. All of the aforementioned mutations
can be made on the background of
wt IL-2, or
wt IL-2 with the substitution C125S, which is a substitution that promotes IL-2 stability
by eliminating an unpaired cysteine residue. This invention can also be used with
other mutations or truncations that improve production or stability without significantly
impacting IL-2 receptor activating activity.
[0045] The variants of this invention optionally include conservatively substituted variants
that apply to both amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to those nucleic acids
which encode identical or essentially identical amino acid sequences, or where the
nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
Specifically, degenerate codon substitutions may be achieved by generating sequences
in which the third position of one or more selected (or all) codons is substituted
with mixed base and/or deoxyinosine residues (
Batzer et al., Nucleic Acid Res. 19:5081 (1991);
Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985);
Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the codons GCA, GCC,
GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine
is specified by a codon, the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic acid variations are
silent variations, which are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize that each codon
in a nucleic acid (except AUG, which is ordinarily the only codon for methionine,
and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described sequence.
[0046] With regard to conservative substitution of amino acid sequences, one of skill will
recognize that individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino
acid or a small percentage of amino acids in the encoded sequence is a conservatively
modified variant where the alteration results in the substitution of an amino acid
with a chemically similar amino acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively modified variants
are in addition to and do not exclude polymorphic variants, interspecies homologs,
and alleles of the invention.
[0047] The following groups each contain amino acids that are conservative substitutions
for one another:
- 1) Alanine (A), Glycine (G);
- 2) Serine (S), Threonine (T);
- 3) Aspartic acid (D), Glutamic acid (E);
- 4) Asparagine (N), Glutamine (Q);
- 5) Cysteine (C), Methionine (M);
- 6) Arginine (R), Lysine (K), Histidine (H);
- 7) Isoleucine (I), Leucine (L), Valine (V); and
- 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
THE FC PROTEIN MOIETY
[0048] A key design choice is the nature of the Fc protein moiety. The main therapeutic
applications of Fc fusion proteins are (1) endowing the fusion partner protein with
immunoglobulin Fc effector functions; or (2) increasing the circulating half-life
of the fusion partner protein (
Czajkowsky, et al., 2012, EMBO Mol Med. 4:1015-28). The primary effector functions of IgG proteins are Complement-Dependent Cytotoxicity
(CDC) and Antibody-Dependent Cellular Cytotoxicity (ADCC), functions mediated by Fc
binding to complement protein C1q and to IgG-Fc receptors (FcγR), respectively. These
effector functions are important when the therapeutic protein is used to direct or
enhance the immune response to a particular antigen target or cell. The fusion protein
of this invention is designed solely to increase the circulating half-life of the
IL2 Selective Agonist moiety, and effector functions are not needed and can even be
toxic, and thus expressly not desired. For instance, an IL2 Selective Agonist-Fc fusion
protein with an effector function-competent Fc can potentially kill the Treg cells
that the fusion protein of this invention is seeking to activate and expand, exactly
the opposite of the therapeutic goal for autoimmune diseases. There are four human
IgG subclasses which differ in effector functions (CDC, ADCC), circulating half-life,
and stability (
Salfeld, J. G., 2007, Nature Biotechnology 25:1369 -72). IgG1 possesses Fc effector functions, is the most abundant IgG subclass, and is
the most commonly used subclass in US FDA-approved therapeutic proteins. IgG2 is deficient
in Fc effector functions, but is subject to dimerization with other IgG2 molecules,
and is also subject to instability due to scrambling of disulfide bonds in the hinge
region. IgG3 possesses Fc effector functions, and has an extremely long, rigid hinge
region. IgG4 is deficient in Fc effector functions, has a shorter circulating half-life
than the other subclasses, and the IgG4 dimer is biochemically unstable due to only
a single disulfide bond in the hinge region leading to the exchange of H chains between
different IgG4 molecules. A skilled artisan would recognize that Fc protein moieties
from IgG2 and IgG4 do not possess effector functions and can be used in this invention.
The skilled artisan would also recognize that Fc sequence modifications have been
described in the art that such that the hinge region of IgG2 Fc can be modified to
prevent aggregation, or that the hinge region of IgG4 Fc can be modified to stabilize
dimers. Alternatively, effector function-deficient variants of IgG1 have been generated.
One such variant has an amino acid substitution at position N297, the location of
an N-linked glycosylation site. Substitution of this asparagine residue removes the
glycosylation site and significantly reduces ADCC and CDC activity (
Tao, M. H., et al., 1989, J Immunol. 143:2595-2601). This variant is used as an exemplary case in the invention herein. Another effector
function deficient IgG1 variant is IgG1(L234FAL235E/P331S) (
Oganesyan, et al., 2008, Acta Crystallogr D Biol Crystallogr. 64:700-4), which mutates amino acids in the C1q and FcγR binding sites, and one skilled in
the art would consider using these or similar Fc variants to generate effector-deficient
and stable IL2SA-Fc fusion proteins. A skilled artisan would also recognize that forms
of Fc protein moieties engineered to be stable monomers rather than dimers (
Dumont, J. A., et., al., 2006, BioDrugs 20:151-60;
Liu Z, et al., J Biol Chem. 2015 20;290:7535-62) can also be combined with the IL-2 selective agonist of this invention. In addition,
a skilled artisan would recognize that a functionally monomeric heterodimer composed
of an IL-2-Fc H chain polypeptide combined with an Fc H chain polypeptide and assembled
using bispecific antibody technology (
Zhu Z, et al., 1997 Protein Sci. 6:781-8) can also be combined with the IL-2 Selective Agonist of this invention. Some IL-2
Fc fusion proteins have been made with intact IgG antibody molecules, either with
(
Penichet, M. L., et., al.,1997, Hum Antibodies. 8:106-18) or without (
Bell, et al., 2015, J Autoimmun. 56:66-80) antigen specificity in the IgG moiety. In addition, a skilled artisan will recognize
that Fc variants that lack some or all of the hinge region can be used with this invention.
[0049] Fc fusion proteins can be made in two configurations, indicated here as X-Fc and
Fc-X, where X, the fusion partner protein, is at the N-terminus and Fc is at the C-terminus,
and Fc-X, where the Fc is at the N-terminus, and fusion partner protein is at the
C-terminus (FIGURE 2). There are examples in the literature showing that different
fusion partners can have distinct preferences for N- or C-terminal Fc fusions. For
instance, FGF21 has been shown to have a strong preference for the Fc-X configuration.
Fc-FGF21 has receptor-activating bioactivity essentially the same as FGF21 itself,
while FGF21-Fc has 1000-fold reduced bioactivity (
Hecht, et al., 2012, PLoS One. 7(11):e49345). A number of IL-2 Fc fusion proteins have been made for various applications, and
these have been reported to have good IL-2 bioactivity when directly fused to Fc in
both the Fc-X (
Gillies, et al., 1992, Proc Natl Acad Sci, 89:1428-32;
Bell, et al., 2015, J Autoimmun. 56:66-80) and X-Fc (
Zheng, X. X., et al., 1999, J Immunol. 163:4041-8) configurations.
Gavin, et al. (US 20140286898 A1) describes Fc fusion proteins containing IL-2 and certain IL-2 variants in the in
the Fc-X configuration that have bioactivity similar to that of the free IL-2 cytokine,
but in contrast to the results of Zheng et al. (
Zheng, X. X., et al., 1999, J Immunol. 1999, 163:4041-8) found that IL-2 variant fusion proteins in the X-Fc configuration have reduced or
no bioactivity. Thus, Gavin, et al. generally teaches away from N-terminal IL-2 Fc
fusion proteins. Another factor that influences the choice of fusion protein configuration
is the impact on circulating half-life. A recurring finding in the literature is that
IL-2 fusion proteins in the Fc-X configuration have relatively low circulating half-lives,
much less than the 21 day half-life of human IgG1 in humans or the half-lives of current
FDA-approved Fc fusion proteins (TABLE I). IgG-IL2 fusion proteins in the Fc-X configuration
have been reported to have relatively short circulating half-lives on the order of
hours in mice (
Gillies S. D., 2002 Clin Cancer Res., 8:210-6;
Gillies, S. D., US 2007/0036752 A2;
Bell C. J., 2015 J Autoimmun. 56:66-80) and on the order of 3.3 hours (
Ribas A., J 2009 Transl Med. 7:68) and 3.7 hours (
King D. M., 2004 J Clin Oncol., 22:4463-73) in humans, and Fc-IL2 fusion proteins have been reported to have circulating half-lives
of 12.5 hours in mice (
Zhu E. F., Cancer Cell. 2015, 13;27(4):489-501). Proteolysis between the C-terminus of the Fc moiety and the IL-2 moiety contributes
to the short circulating half-lives (
Gillies S. D., 2002 Clin Cancer Res., 8:210-6;
Zhu E. F., 2015 Cancer Cell. 27:489-501). Because of these relatively short half-lives, we have focused on IL2 Selective
Agonist Fc fusion proteins in the X-Fc configuration.
LINKER
[0050] The amino acid sequence at the junction between the Fc and the fusion partner protein
can be either (1) a direct fusion of the two protein sequences or (2) a fusion with
an intervening linker peptide. Of the 10 Fc fusion proteins that are presently approved
by the US FDA for clinical use (TABLE I), 8 are direct fusions of the fusion partner
protein with Fc, while 2 possess linker peptides, so many Fc fusion proteins can be
functional without linker peptides. Linker peptides are included as spacers between
the two protein moieties. Linker peptides can promote proper protein folding and stability
of the component protein moieties, improve protein expression, and enable better bioactivity
of the component protein moieties (
Chen, et al., 2013, Adv Drug Deliv Rev. 65:1357-69). Peptide linkers used in many fusion proteins are designed to be unstructured flexible
peptides. A study of the length, sequence, and conformation of linkers peptides between
independent structural domains in natural proteins has provided a theoretical basis
for the design of flexible peptide linkers (
Argos, 1990, J Mol Biol. 211:943-58). Argos provided the guidance that long flexible linker peptides be composed of small
nonpolar residues like Glycine and small polar resides like Serine and Threonine,
with multiple Glycine residues enabling a highly flexible conformation and Serine
or Threonine providing polar surface area to limit hydrophobic interaction within
the peptide or with the component fusion protein moieties. Many peptide linkers described
in the literature are rich in glycine and serine, such as repeats of the sequence
GGGGS, although an artisan skilled in the art will recognize that other sequences
following the general recommendations of Argos (
Argos, 1990, J Mol Biol. 20;211(4):943-58) can also be used. For instance, one of the proteins described herein is contains
a linker peptide composed of Glycine and Alanine (SEQ ID NO 15). A flexible linker
peptide with a fully extended beta- strand conformation will have an end-to-end length
of approximately 3.5 Å per residue. Thus, a linker peptide of 5, 10, 15, or 20 residues
will have a maximum fully extended length of 17.5 Å, 35 Å, 52.5 Å, or 70 Å, respectively.
The maximal end-to-end length of the peptide linker can also be a guide for defining
the characteristics of a peptide linker in this invention. The goal of a linker peptide
within the current invention is to enable attainment of an appropriate conformation
and orientation of the individual fusion protein moieties to allow the engagement
of the IL-2 Selective Agonist moiety with its cognate receptor and allow the binding
of the Fc moiety to the FcRn to enable fusion protein recycling and a prolonged circulating
half-life. Since the factors influencing these interactions are difficult to predict,
the requirement for and the proper length of a linker peptide must be empirically
tested and determined. Many Fc fusion proteins do not require linker peptides, as
evidenced by the 8 out of 10 US FDA-approved Fc fusion proteins lacking such peptides
listed in Table I. In contrast, Dulaglutide, a fusion of GLP-1 and Fc, contains a
15 residue peptide linker which has a strong influence on bioactivity (Glaesner,
US Patent 7,452,966 B2). Prior work in the art on IL-2-Fc fusion proteins indicates that linker peptides
are not necessary for bioactivity. IL-2 fusion proteins containing wt IL-2 or IL-2
with the substitution C125S in the Fc-X orientation have been reported to have IL-2
bioactivity similar to that of the free IL-2 cytokine without (
Gillies, et al., 1992, Proc Natl Acad Sci, 89:1428-32;
Gavin, et al., US Patent Application 20140286898 A1) or with (
Bell, et al., 2015, J Autoimmun. 56:66-80) peptide linkers. In the X-Fc orientation, Zheng et al. reported IL-2 bioactivity
of an IL-2 fusion protein in the X-Fc configuration that was essentially indistinguishable
from that of IL-2 itself (
Zheng, X. X., et al., 1999, J Immunol. 1999, 163:4041-8). This extensive prior art teaches that fusion of an IL-2 protein with Fc will not
require a linker peptide in order to have high IL-2 bioactivity. However, Gavin et
al. reported that Fc fusion proteins in the X-Fc configuration containing certain
IL-2 variants with altered receptor selectivity have reduced or no bioactivity either
without a peptide linker or with a 5 residue peptide linker (
Gavin, et al., US Patent Application 20140286898 A1).
BIOASSAYS
[0051] Robust and quantitative bioassays are necessary for the characterization of the biological
activity of candidate proteins. These assays should measure the activation of the
IL2 receptor, measure the downstream functional consequences of activation in Tregs,
and measure therapeutically-relevant outcomes and functions of the activated Tregs.
These assays can be used the measure the therapeutic activity and potency of IL2 Selective
Agonist molecules, and can also be used for measurement of the pharmacodynamics of
an IL2 Selective Agonist in animals or in humans. One assay measures the phosphorylation
of the signal transduction protein STAT5, measured flow cytometry with an antibody
specific for the phosphorylated protein (pSTAT5). Phosphorylation of STAT5 is an essential
step in the IL-2 signal transduction pathway. STAT5 is essential for Treg development,
and a constitutively activated form of STAT5 expressed in CD4+CD25+ cells is sufficient
for the production of Treg cells in the absence of IL-2 (
Mahmud, S. A., et al., 2013, JAKSTAT 2:e23154). Therefore, measurement of phosphorylated STAT5 (pSTAT5) in Treg cells will be recognized
by someone skilled in the art as reflective of IL-2 activation in these cells, and
will be predictive of other biological outcomes of IL-2 treatment given appropriate
exposure time and conditions. Another assay for functional activation measures IL-2-stimulated
proliferation of Treg cells. Someone skilled in the art will recognize that Treg proliferation
can be measured by tritiated thymidine incorporation into purified Treg cells, by
an increase in Treg cell numbers in a mixed population of cells measured by flow cytometry
and the frequencies of CD4+CD25+FOXP3+ or the CD4+CD25+CD127- marker phenotypes, by
increased expression in Treg cells of proliferation-associated cell cycle proteins,
such as Ki-67, or by measurement of the cell division-associated dilution of a vital
fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE) by flow cytometry
in Treg cells. Another assay for functional activation of Tregs with IL-2 is the increased
stability of Tregs. pTreg cells are thought by some to be unstable, and have the potential
to differentiate into Th1 and Th17 effector T cells. IL-2 activation of Tregs can
stabilize Tregs and prevent this differentiation (
Chen, Q., et al., 2011, J Immunol. 186:6329-37). Another outcome of IL-2 stimulation of Tregs is the stimulation of the level of
Treg functional effector molecules, such as CTLA4, GITR, LAG3, TIGIT, IL-10, CD39,
and CD73, which contribute to the immunosuppressive activity of Tregs.
[0052] To develop an IL2 Selective Agonist Fc protein, we initially focused on proteins
in the X-Fc configuration because of the short circulating half-lives that have been
reported for IL-2 fusion proteins in the Fc-X configuration. The first two proteins
produced and tested, one with and one without a linker peptide, unexpectedly showed
that the protein with the peptide linker had IL-2 bioactivity and that the protein
without the peptide linker had no detectable bioactivity. Both proteins exhibited
high binding affinity for IL2RA, indicating that both proteins were properly folded.
These results suggested that a linker peptide was necessary for IL-2 receptor activation
and bioactivity. A series of additional analogs was then produced to eliminate other
variables and to test this hypothesis. The results from these studies led to the discovery
of key structure-activity relationships for this therapeutic protein and created novel
molecules with the desired activity and pharmaceutical attributes.
FORMULATION
[0053] Pharmaceutical compositions of the fusion proteins of the present invention are defined
as formulated for parenteral (particularly intravenous or subcutaneous) delivery according
to conventional methods. In general, pharmaceutical formulations will include fusion
proteins of the present invention in combination with a pharmaceutically acceptable
vehicle, such as saline, buffered saline, 5% dextrose in water, or the like. Formulations
may further include one or more excipients, preservatives, solubilizers, buffering
agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation
are well known in the art and are disclosed, for example, in
Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,
Easton, Pa., 19.sup.th ed., 1995.
[0054] As an illustration, pharmaceutical formulations may be supplied as a kit comprising
a container that comprises fusion proteins of the present invention. Therapeutic proteins
can be provided in the form of an injectable solution for single or multiple doses,
as a sterile powder that will be reconstituted before injection, or as a prefilled
syringe. Such a kit may further comprise written information on indications and usage
of the pharmaceutical composition. Moreover, such information may include a statement
that the fusion proteins of the present invention is contraindicated in patients with
known hypersensitivity to fusion proteins of the present invention.
[0055] The IL-2 selective agonist fusion proteins of this invention can be incorporated
into compositions, including pharmaceutical compositions. Such compositions typically
include the protein and a pharmaceutically acceptable carrier. As used herein, the
term "pharmaceutically acceptable carrier" includes, but is not limited to, saline,
solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Supplementary active compounds (e.g., antibiotics) can also be incorporated into the
compositions.
[0056] A pharmaceutical composition is formulated to be compatible with its intended route
of administration. The IL-2 selective agonist fusion proteins of the invention is
likely that to be administered through a parenteral route. Examples of parenteral
routes of administration include, for example, intravenous, intradermal, and subcutaneous.
Solutions or suspensions used for parenteral application can include the following
components: a sterile diluent such as water for injection, saline solution, polyethylene
glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents
such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such
as acetates, citrates or phosphates and agents for the adjustment of tonicity such
as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono-
and/or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to
a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or plastic.
[0057] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions
or dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable carriers include
physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In
all cases, the composition should be sterile and should be fluid to the extent that
easy syringability exists. It should be stable under the conditions of manufacture
and storage and must be preserved against the contaminating action of microorganisms
such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures thereof. The maintenance
of the required particle size in the case of dispersion may be facilitated by the
use of surfactants, e.g., Polysorbate or Tween. Prevention of the action of microorganisms
can be achieved by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars, polyalcohols such
as mannitol, sorbitol, sodium chloride in the composition.
[0058] Sterile injectable solutions can be prepared by incorporating the active compound
in the required amount in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the active compound into a sterile vehicle, which contains
a basic dispersion medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of sterile injectable solutions,
the preferred methods of preparation are vacuum drying and freeze-drying which yields
a powder of the active ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0059] In one embodiment, the IL-2 selective agonist fusion protein is prepared with carriers
that will protect the IL-2 selective agonist fusion protein against rapid elimination
from the body, such as a controlled release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid. Such formulations can be prepared using standard techniques.
[0060] The pharmaceutical compositions can be included in a container, pack, or dispenser
together with instructions for administration.
ADMINISTRATION
[0061] Fusion proteins of the present invention will preferably be administered by the parenteral
route. The subcutaneous route is the preferred route, but intravenous, intramuscular,
and subdermal administration can also be used. For subcutaneous or intramuscular routes,
depots and depot formulations can be used. For certain diseases specialized routes
of administration can be used. For instance, for inflammatory eye diseases intraocular
injection can be used. Fusion proteins can be used in a concentration of about 0.1
to 10 mcg/ml of total volume, although concentrations in the range of 0.01 mcg/ml
to 100 mcg/ml may be used.
[0062] Determination of dose is within the level of ordinary skill in the art Dosing is
daily or weekly over the period of treatment, or may be at another intermittent frequency.
Intravenous administration will be by bolus injection or infusion over a typical period
of one to several hours. Sustained release formulations can also be employed. In general,
a therapeutically effective amount of fusion proteins of the present invention is
an amount sufficient to produce a clinically significant change in the treated condition,
such as a clinically significant change in circulating Treg cells, a clinically significant
change in Treg cells present within a diseased tissue, or a clinically significant
change in a disease symptom.
[0063] The data obtained from the cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such compounds lies
preferably within a range of circulating concentrations that include the half maximal
effective concentration (EC50; i.e., the concentration of the test compound which
achieves a half-maximal stimulation of Treg cells) with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed and the
route of administration utilized. For any compound used in the method of the invention,
the therapeutically effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating plasma concentration
range that includes the EC50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in plasma may be
measured, for example, by enzyme-linked immunosorbent assays.
[0064] As defined herein, a therapeutically effective amount of a IL-2 selective agonist
fusion protein (i.e., an effective dosage) depends on the polypeptide selected and
the dose frequency. For instance, single dose amounts in the range of approximately
0.001 to 0.1 mg/kg of patient body weight can be administered; in some embodiments,
about 0.005, 0.01, 0.05 mg/kg may be administered. The compositions can be administered
from one time per day to one or more times per week, or one or more times per month;
including once every other day. The skilled artisan will appreciate that certain factors
may influence the dosage and timing required to effectively treat a subject, including
but not limited to the severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, the level of Treg cells present in the patient,
and other diseases present Moreover, treatment of a subject with a therapeutically
effective amount of the IL-2 selective agonist fusion protein of the invention is
likely to be a series of treatments.
Autoimmune Diseases
[0065] Some of the diseases that can benefit from the therapy of this invention have been
noted. However, the role of Treg cells in autoimmune diseases is a very active area
of research, and additional diseases will likely be identified as treatable by this
invention. Autoimmune diseases are defined as human diseases in which the immune system
attacks its own proteins, cells, and tissues. A comprehensive listing and review of
autoimmune diseases can be found in
The Autoimmune Diseases (Rose and Mackay, 2014, Academic Press).
Other fusion proteins
[0066] Because the purpose of the Fc protein moiety in this invention is solely to increase
circulating half-life, one skilled in the art will recognize that the IL-2 selective
agonist moiety could be fused to the N-terminus of other proteins to achieve the same
goal of increasing molecular size and reducing the rate of renal clearance, using
the structure-activity relationships discovered in this invention. The IL2 selective
agonist could be fused to the N-terminus of serum albumin (
Sleep, D., et al., 2013, Biochim Biophys Acta. 1830:5526-34), which both increases the hydrodynamic radius of the fusion protein relative to
the IL-2 moiety and is also recycled by the FcRN. A skilled artisan would also recognize
that the IL2 selective agonist moiety of this invention could also be used to the
N-terminus of recombinant non-immunogenic amino acid polymers. Two examples of non-immunogenic
amino acid polymers developed for this purpose are XTEN polymers, chains of A, E,
G, P, S, and T amino acids (
Schellenberger, V., et al., 2009, Nat Biotechnol. 27:1186-90), and PAS polymers, chains of P, A, and S amino acid residues (
Schlapschy, M., et. al., 2007, Protein Eng Des Sel. 20:273-84).
EXAMPLES
[0067] The following examples are provided by way of illustration only and not by way of
limitation. Those of skill will readily recognize a variety of noncritical parameters
which could be changed or modified to yield essentially similar results.
Example 1. Cloning, expression, and purification of IL-2 selective agonist-IgG Fc fusion proteins
[0068] A cDNA encoding N88RL9AG1 (SEQ ID NO 4) was constructed by DNA synthesis and PCR
assembly. The N88RL9AG1 construct was composed of the mouse IgG1 signal sequence,
the mature human IL-2 (SEQ ID NO 1) sequence with the substitutions N88R and C125S,
a 9 amino acid linker peptide sequence (SEQ ID NO 15), and the Fc region of human
IgG1 containing the substitution N297A (SEQ ID NO 2). N88R/IL2 is an IL2 selective
agonist with reduced binding to IL2RB and selective agonist activity on IL2Rαβγ receptor-expressing
cells (
Shanafelt, A. B., et al., 2000, Nat Biotechnol. 18:1197-202). Elimination of the N-linked glycosylation site at N297 on IgG1 Fc reduces Fc effector
functions (
Tao, M. H., et al., 1989, J Immunol. 143:2595-2601). D20HLOG2 was composed of the mouse IgG1 signal sequence, IL-2 (SEQ ID NO 1) with
the substitutions D20H and C125S, and an Fc protein moiety derived from human IgG2
(SEQ ID NO 3). The D20H IL-2 variant has been reported to possess selective agonist
activity similar to N88R (
Cassell, D. J., et al., 2002, Curr Pharm Des., 8:2171-83).
[0069] These cDNAs were cloned into pcDNA3.1(+) (Life Technologies, Carlsbad, CA) using
the restriction sites HindIII and NotI. Purified expression vector plasmid containing
the construct was transiently transfected into HEK293 cells. HEK293 cells were seeded
into a shake flask 24 hours before transfection, and were grown using serum-free chemically
defined media. The DNA expression constructs were transiently transfected into 0.1
liter of suspension HEK293 cells. After 24 hours, cells were counted to obtain the
viability and viable cell count The cultures were harvested at day 5 and the conditioned
media supernatant was clarified by centrifugation at 3000 X g for 15 minutes. The
protein was purified by running the supernatant over a Protein A column (GE Healthcare),
eluting with 0.25% acetic acid (pH 3.5), neutralizing the eluted protein with 1M Tris
(pH 8.0), and dialyzing against 30 mM HEPES, pH 7,150 mM NaCI. The samples were then
sterile filtered through a 0.2 µm membrane filter and analyzed by SDS PAGE under reducing
and nonreducing conditions. The proteins migrated as a disulfide-linked dimer. Protein
concentration determined by absorbance using the calculated extinction coefficient
of 1.11 mg/ml cm
-1, and aliquots stored at -80C.
[0070] The cytokines N88R/IL2 and D20H/IL2 are variants of SEQ ID NO 1 and were produced
in E coli essentially as described in
US patent 6,955,807 B1, except for the addition of the additional mutation C125S for improved stability.
Example 2. Determination of receptor-binding activity of N88RL9AG1 and D20HL0G2.
[0071] To determine if N88RL9AG1 and D20HL0G2 were properly folded, their affinity to the
IL-2 receptor subunits IL2RA and IL2RB was determined by surface plasmon resonance
(SPR) using a Biacore T-200 instrument (GE Healthcare). IL2RA and IL2RB extracellular
domain proteins and IL-2 protein (R&D Systems, Minneapolis, MN) were immobilized on
CM-5 Biacore chips by NHS/EDC coupling to final RU (resonance units) values of 30
and 484, respectively. The kinetics of binding to IL2RA was measured at five concentrations
of IL2 and N88RL9AG1 ranging from 0.6 nM to 45 nM at a flow rate of 50 ul/minute.
The kinetics of binding to IL2RB was measured at five concentrations ranging from
16.7 nM to 450 nM for IL2 and from 14 nM to 372 nM for the Fc fusion proteins at a
flow rate of 10 ul/minute. The dissociation constants (Kd) were calculated from the
kinetic constants using the Biacore evaluation software version 2.0, assuming 1:1
fit for IL-2 and the bivalent fit for N88RL9AG1 and D20HL0G2. Equilibrium Kd values
were calculated by the Biacore evaluation software using steady-state binding values.
[0072] Binding to IL2RA was detected for both IL-2 and N88RL9AG1. The Rmax value for N88RL9AG1,
14.43, was 5.5 fold higher than that of IL2, 2.62, consistent with the fact that N88RL9AG1
(82,916 g/M) has a greater molecular weight than IL-2 (15,444 g/M). The kon, koff,
and Kd values for IL-2 were in the range expected from published SPR values (Table
II). The affinity of N88RL9AG1 was approximately 2-fold greater than that of IL2 as
determined by both the kinetic and equilibrium methods. Binding of IL2 to IL2RB was
detected with an Rmax of 6.19. The values determined for kon, koff, and Kd are within
the range reported in the literature. Reported values are 3.1 X 10
-8 M (IL2RA) and 5.0 X 10
-7M (IL2RB) (
Myszka, D. G., et al., 1996, Protein Sci. 5:2468-78); 5.4 X 10
-8 M (IL2RA) and 4.5 X 10
-7 (IL2RB) (
Shanafelt, A. B., et al., 2000, Nat Biotechnol.18:1197-202); and 6.6 X 10
-9 M (IL2RA) and 2.8 X 10
-7 M (IL2RB) (
Ring, A. M., et al., 2012, Nat Immunol. 13:1187-95). Essentially no binding of N88RL9AG1 to IL2RB was detected, with a slight binding
detected at the highest concentration tested (Rmax = 0.06), far below that expected
based on the molecular weight difference between IL2 and N88RL9AG1 and based on the
IL2RA binding results. The D20HL0G2 protein was also tested for binding to IL2RA,
and was found to have a Kd of 8.3 X 10
-9 M, similar to that of N88RL9AG1. Thus, SPR binding studies indicated that both N88RL9AG1
and D20HL0G2 proteins bind to IL2RA, indicating that the proteins are properly folded.
Example 3. Bioactivity of N88RL9AG1 and D20HL0G2 on T cells.
[0073] The bioactivity of N88RL9AG 1 and D20HL0G2 on T cells was determined by measuring
phosphorylated STAT5 (pSTAT5) levels in specific T cell subsets. Levels of pSTAT5
were measured by flow cytometry in fixed and permeabilized cells using an antibody
to a phosphorylated STAT5 peptide. Treg cells constitutively express CD25, and cells
that are in the top 1% of CD25 expression levels are highly enriched for Treg cells
(
Jailwala, P., et al., 2009, PLoS One. 2009; 4:e6527;
Long, S. A., et al., 2010, Diabetes 59:407-15). Therefore, the flow cytometry data was gated into CD25
high (the top 1-2% of CD25 expressing cells) and CD25
-/low groups for the Treg and CD4 effector T cell subsets, respectively.
[0074] Cryopreserved CD4+ T cells (Astarte Biologies, Seattle, WA) were defrosted, washed
in X-VIVO 15 (Lonza, Allendale, NJ) media containing 1% human AB serum (Mediatech,
Manassas, VA) and allowed to recover for 2 hours at 37 C. Cells were then distributed
in 0.1 ml into 15x75 mm tubes at a concentration of 5 x 10
6 cells/ml. Cells were treated with varying concentrations of IL-2 or Fc fusion proteins
for 10 minutes at 37 C. Cells were then fixed with Cytofix Fixation Buffer at 37C
for 10 minutes, permeabilized with Perm Buffer III (BD Biosciences, Santa Clara, CA)
for 30 minutes on ice, and then washed. Cells were then stained with a mixture of
anti-CD4-Pacific Blue (BD Biosciences, Santa Clara, CA), anti-CD25-AF488 (eBioscience,
San Diego, CA), and anti-pSTAT5-AF547 (BD Biosciences) antibodies at concentrations
recommended by the manufacturer for 30 minutes at 20 C, washed, and flow cytometry
data acquired on an LSRII instrument (BD Biosciences). Data was analyzed using Flowjo
analysis software (Flowjo, Ashland, OR).
[0075] The results with N88RL9AG1 in this assay indicated that compared to IL-2 N88RL9AG1
had remarkable selectivity for the Treg population (FIGURE 3A). N88RL9AG1 activated
less than 1% of CD4+ cells, with very strong selectivity for CD25
high cells. In contrast, IL-2 activated over 80% of CD4+ T cells at a concentration of
40 nM, with a high proportion of the pSTAT5+ cells expressing low levels or no CD25.
Even at 4 pM, the lowest concentration tested, IL-2 still stimulated significant pSTAT5
levels in both CD25
-/low cells and CD25
high cells.
[0076] D20HL0G2 was then tested for activity in the CD4+ T cell pSTAT5 assay. Unexpectedly,
D20HL0G2 had no activity in this assay (FIGURE 3B). An additional control with 10
-8 M D20H/IL2 cytokine (the variant IL-2 cytokine not fused to Fc) showed robust and
selective pSTAT5 activation of CD25
high cells (FIGURE 3C). The lack of activity with D2UHL0G2 was especially surprising given
that D20HL0G2 bound to IL2RA with a Kd similar to that of IL-2 and N88RL9AG1, indicating
it was properly folded.
[0077] To confirm that the CD25
high cells selectively activated by N88RL9AG1 were Tregs, activated cells were co-stained
for both pSTAT5 and FOXP3, another molecular marker for Treg cells. CD4+ cells were
treated with 4 nM IL-2 or N88RL9AG1, fixed, and permeabilized as described above for
pSTAT5 staining, and then were subsequently treated with 1 ml FOXP3 Perm Buffer (BioLegend,
San Diego, CA) for 30 min at room temperature, and then washed and resuspended in
FOXP3 Perm Buffer. Permeabilized cells were stained with a mixture of anti-FOXP3-eFluor450,
anti-CD25-AF488 (eBioscience, San Diego, CA), and anti-pSTAT5-AF547 (BD Biosciences)
antibodies for 30 minutes at 20 C, washed, and analyzed by flow cytometry. The results
of this experiment indicated that a high proportion of N88RL9AG1-treated cells with
activated STAT5 (pSTAT5+ cells) were also expressing high levels of FOXP3. This result
provides further evidence that the activated cells are highly enriched for Treg cells.
In contrast, IL-2 treated pSTAT5+ cells were both FOXP3+ and FOXP3-, with the majority
being FOXP3- cells.
Example 4. Determination of structure-activity relationships important for bioactivity.
[0078] The unexpected results described in Example 3 suggested that the IL2 bioactivity
detected with N88RL9AG1 but not with D20HL0G2 was due to the presence of a linker
peptide. To verify this finding and to eliminate the contribution of other variables,
such as the isotype of the Fc moiety and the selectivity mutation in the IL-2 moiety,
a panel of analogs, all using the IgG1 N297A Fc, were designed and produced (TABLE
III).
[0079] cDNAs were constructed and proteins expressed and purified as described in Example
1, except that the C-terminal Lysine residue of the Fc was deleted in all constructs
and that the production cell cultures were in a volume of 30 ml instead of 100 ml.
All proteins were recovered in good yield. In fact, comparison of the yields of the
N88R/IL2 series of molecules indicated a clear trend of increasing protein yield with
increasing peptide linker length, with N88RL20AG1 (with the longest peptide linker)
recovery 6.8 fold higher than N88RLOAG1 (with no peptide linker) (FIGURE 5A). The
basis for the increased yields of linker peptide-containing proteins is not yet clear,
but could be due to increased expression level, increased secretion rate, increased
protein stability, or increased purification efficiency. Interestingly, the yield
of WTL15AG1 was only marginally higher (1.8 fold) than that of WTL0AG1, compared to
a 4.5 fold higher yield of N88RL15AG1 compared to N88RL0AG1. D20HL15AG1 yield was
similar to N88RL15AG1 yield, indicating the IL-2 selectivity mutation has no significant
effect on yield, and both of these proteins had significantly higher yields (4.3 fold
and 3.4 fold, respectively) than AG1L15D20H (FIGURE 5B). Collectively, these results
indicated that increasing peptide linker length was associated with higher protein
yield of N88R/IL2 containing Fc fusion proteins, that the yield of Fc fusion proteins
containing
wt IL-2 was much less sensitive to the presence of a linker peptide, and IL-2-Fc fusion
proteins in the X-Fc configuration are produced
[0080] These purified proteins were tested in a human T cell pSTAT5 bioassay essentially
as described in Example 3, except that human CD3+ T cells (negatively selected) were
used instead of CD4+ cells, and the cells were incubated with test proteins for 20
min rather than 10 min.
[0081] The results from the N88R/IL2 series of molecules showed that bioactivity in the
Treg-enriched population was dramatically influenced by peptide linker length (FIGURE
6A). The pSTAT5 signal (% pSTAT5+ cells) in the Treg population increased progressively
with increasing peptide linker length. This increased bioactivity was reflected both
in the maximal pSTAT5 signal at 10
-8 M test protein and by the EC50 values (TABLE IV). N88RL20AG1, the protein with the
longest peptide linker, showed a 4.2 fold increase in the maximal pSTAT5 signal over
N88RL0AG1. Because the N88RLOAG1 pSTAT5 signal did not reach 50% of IL-2 activation
at its highest concentration (10
-8 M), it was not possible to determine fold improvement in EC50 of the proteins containing
linker peptides over N88RL0AG1. However, based on N88RL20AG1 EC50 and the highest
concentration of N88RL0AG1 tested, it can be estimated that N88RL20AG1 will exhibit
a >100 fold lower EC50 than N88RL0AG1.
[0082] As expected, there was essentially no detectable activity of any of the N88R/IL2
molecules on the CD25
-/low population, while 10
-8 M IL-2 stimulated pSTAT5 activity in 54.2 % of the CD25
-/low cells (Figure 6B).
[0083] The comparison of WTL0AG1 and WTL15AG1 showed that linker peptides have a much less
significant effect on
wt IL-2-Fc fusion proteins than N88R/IL2-Fc fusion proteins (FIGURE 7). In the Treg
subpopulation, both WTL0AG1 and WTL15AG1 had significant bioactivity, and in fact
stimulated an approximately 2-fold higher maximum level of pSTAT5 phosphorylation
than IL-2. However, WTL0AG1 and WTL15AG1 also stimulated large pSTAT5 signals in CD25
-/low cells at an approximately 10 fold higher concentration. WTL15AG1 and WTLOAG1 exhibited
an approximately 10 fold difference in EC50 values in both the Treg and the CD25
-/low cell populations.
[0084] The maximum pSTAT5 signal of D20HL 15AG1 in Tregs was significantly less than that
of N88RL15AG1 (FIGURE 8). This suggests that the lack of any detectable activity in
Example 3 with D20HL0G2 was due in part to a lower activity of the D20H/1L2 moiety
in the context of an Fc fusion protein compared to the N88R/IL2 moiety. The activity
of AG1L15D20H was slightly higher than that of D20HL15AG1, indicating that the configuration
of the IL-2 moiety in the Fc fusion protein (ie., X-Fc vs Fc-X) did not have a major
effect on Treg bioactivity.
[0085] Collectively, these results define key features of N88R/IL2-Fc fusion proteins necessary
for optimal bioactivity. N88R/IL2-Fc proteins require a linker peptide for optimal
Treg bioactivity, with a trend of increasing bioactivity with increasing linker peptide
length. Second, in line with the work of others, linker peptides have a more modest
effect on the bioactivity of Fc fusion proteins containing
wt IL-2. These differing requirements for a linker peptide may a consequence of the
fact that N88R/IL2 is deficient in binding to IL2RB, which could possibly result in
more stringent requirements for receptor engagement and increasing the sensitivity
to steric hinderance from the Fc fusion protein partner. These results also define
the most potent IL2 Selective Agonist-Fc fusion proteins.
Example 5. Selectivity of IL2 SelectiveAgonist-Fc fusion proteins in human PBMC
[0086] To determine the selectivity of N88R/IL2-Fc fusion proteins in a broader biological
context, an assay was developed to measure STATS activation across all key immune
cell types in crude unfractionated human PBMC. Human PBMC were isolated by Ficoll-Hypaque
centrifugation from a normal volunteer. 10
6 PBMC were suspended in X-VIVO15 media with glucose (Lonza) and 10% FBS (Omega), and
were treated with 10
-8 M test proteins for 20 min at 37°C. Cells were then treated with Foxp3/Transcription
Factor Staining Buffer Set (EBIO) according to the manufacturers instructions. Cells
were then fixed with Cytofix buffer and permeabilized with Perm Buffer III as described
in Example 3. Fixed and permeabilized cells were then washed with 1% FBS/PBS and stained
with antibody mixture for 60 minutes at room temperature in the dark. Stained cells
were then washed in 1% FBS/PBS, resuspended in PBS, and analyzed on a Fortessa flow
cytometer (BD Biosciences). The antibody mix consisted of: anti-CD4-PerCP-Cy5.5 (BD,
#560650), anti-pSTAT5-AF-488 (BD, #612598), anti-CD25-PE (BD, #560989), anti-CD56-PE-CF594
(BD, #562328), anti-FOXP3-AF647 (BD, #560889), anti-CD3-V450 (BD, 560366), and anti-CD8-BV650
(Biolegend, #301041). This staining procedure enabled monitoring of pSTAT5 levels
in 7 key immune cells types.
[0087] Cell phenotypes were defined as follows: Treg cells: CD3+, CD4+, Foxp3+, CD25
high, CD8-, CD56-; activated CD4 Teff cells: CD3+, CD4+, Foxp3-, CD25
high, CD8-, CD56-; CD4 Teff cells: CD3+, CD4+, Foxp3-, CD25
low, CD8-, CD56-; NKT cells: CD3+, CD4-, Foxp3-, CD25
low, CD8-, CD56+; NK cells: CD3-, CD4-, Foxp3-, CD25
low, CD8-, CD56+; B cells: CD3-, CD4-, Foxp3-, CD25
low, CD8-, CD56-.
[0088] Proteins were tested in this assay at a concentration of 10
-8 M. The results, shown in FIGURE 9 and summarized in TABLE V, show that N88RL15AG1
exhibited remarkable selectivity compared to
wt IL2 and WTL15AG1, both of which activated pSTAT5 in large fractions of all the cell
populations. N88RL15AG1 stimulated pSTAT5 signal in the Treg population at close to
the level of
wt IL-2, with insignificant activation of the other cell types with the exception of
NK cells. Additional analysis (not shown) showed that the pSTAT5+ NK cells were CD25
high, which is characteristic of NK-CD56
bright cells, an NK cell subpopulation which also has immunoregulatory activity (
Poli, A, et al., 2009 Immunology. 126(4):458-65). Several cell types that had low-level pSTAT5 signals with N88R/IL2 (activated CD4
Teff cells, CD4 Teff cells, NK T cells, and NK cells) exhibited no or lower pSTAT5
signals with N88RL15AG1. These results demonstrate the activity and high selectivity
of N88RL15AG1 for Tregs in a complex biological milieu.
TABLES
TABLE I. US FDA-approved Fc fusion proteins and their characteristics
[0089]
TABLEI
DRUG |
Fc Isotype |
Fusion Partner |
N vs C fusion |
Linker Peptide |
Half-life (days) |
Romiplostim |
G1 |
TPO-R peptide |
C |
Y |
3.5 |
Etanercept |
G1 |
P75 TNFa-R |
N |
N |
4.3 |
Alefacept |
G1 |
LFA3 |
N |
N |
10.1 |
Rilonacept |
G1 |
IL1-R |
N |
N |
8.6 |
Abatacept |
G1 |
CTLA4 |
N |
N |
16.7 |
Belatacept |
G1 |
CTLA4 (mut) |
N |
N |
9.8 |
Aflibercept |
G1 |
VEGF R1 + R2 |
N |
N |
n/a |
Dulaglutide |
G4 (mut) |
GLP1 |
N |
Y |
3.7 |
Eloctate |
G1 |
FVIII |
N |
N |
0.8 |
Alprolix |
G1 |
FIX |
N |
N |
3.6 |
Table II. Affinity of IL-2 Fc fusion proteins for IL2RA and IL2RB subunits
[0090]
TABLE II
Ligand |
Analyte |
Method |
kon |
koff |
Kd (M) |
IL2RA |
IL-2 |
Kinetic |
5.85 X 106 |
8.4 X 10-2 |
1.44 X 10-8 |
|
N88RL9AG1 |
Kinetic |
1.78 X 106 |
1.0 X 10-2 |
5.63 X 10-9 |
|
D20HLOG2 |
Kinetic |
1.66 X 107 |
0.137 |
8.30 X 10-9 |
|
IL-2 |
Equilibrium |
- |
- |
1.47 X 10-8 |
|
N88RL9AG1 |
Equilibrium |
- |
- |
9.36 X 10-9 |
IL2RB |
IL-2 |
Kinetic |
5.10 X 105 |
3.0 X 10-1 |
5.87 X 10-7 |
|
N88RL9AG1 |
Kinetic |
nd |
nd |
|
|
IL-2 |
Equilibrium |
- |
- |
2.53 X 10-7 |
|
N88RL9AG1 |
Equilibrium |
- |
- |
7.60 X 10-2 |
TABLE III
Protein |
IL2 |
Peptide Linker |
Configuration |
SEQ ID No |
N88RL0AG1 |
N88R |
0 |
X-Fc |
6 |
N88RL5AG1 |
N88R |
5 |
X-Fc |
7 |
N88RL10AG1 |
N88R |
10 |
X-Fc |
8 |
N88RL15AG1 |
N88R |
15 |
X-Fc |
9 |
N88RL20AG1 |
N88R |
20 |
X-Fc |
10 |
WTL0AG1 |
wt |
0 |
X-Fc |
11 |
WTL15AG1 |
wt |
15 |
X-Fc |
12 |
D20HL15AG1 |
D20H |
15 |
X-Fc |
13 |
AG1L15D20H |
D20H |
15 |
Fc-X |
14 |
TABLE IV
Protein |
EC50 |
Maximal pSTAT5 response at 10-8 M |
Fold increase in maximal pSTAT5 response |
N88RLOAG1 |
>10-8 |
0.33 |
1.0 |
N88KLSAG1 |
>10-8 |
0.52 |
1.6 |
N88KL9AG1 |
7 X 10-10 |
0.96 |
2.9 |
N88RL10AG1 |
9 X 10-10 |
0.90 |
2.7 |
N88RL15AG1 |
4 X 10-10 |
1.22 |
3.7 |
N88RL20AG1 |
1 X 10-10 |
1.40 |
4.2 |
TABLE V
|
Control |
IL-2 |
WTL15AG1 |
N88R/IL2 |
N88RL15AG1 |
Treg cells |
0.8 |
99.9 |
99.8 |
99.9 |
75.1 |
Activated CD4 Teff cells |
0.1 |
70.5 |
65.2 |
3.7 |
0.1 |
CD4 Teff cells |
0.2 |
60.9 |
40.0 |
2.4 |
0.5 |
CD8 Teff cells |
0.1 |
90.2 |
35.4 |
2.3 |
0.1 |
NKT cells |
0.5 |
74.9 |
60.5 |
20.5 |
5.2 |
NK cells |
0.3 |
96.8 |
96.1 |
49.9 |
19.3 |
B cells |
0.1 |
20.9 |
10.6 |
0.2 |
0.1 |
Percentage of pSTAT5+ cells in 7 immune cells types in human PBMC. Cells were treated
with proteins indicated in the column headings and analyzed as described in Example
6. |
SEQUENCE LISTINGS
[0091]
SEQ ID NO.15
>L9
GGGGAGGGG
SEQ ID NO.16
>L5
GGGGS
SEQ ID NO.17
>L10
GGGGSGGGGS
SEQ ID NO.18
>L15
GGGGSGGGGSGGGGS
SEQ ID NO.19
>L20
GGGGSGGGGSGGGGSGGGGS